11C-labeled benzophenone/benzoxazole analogues as an inflammation imaging agent

ABSTRACT

A method of preparing novel [ 11 C]-labeled benzophenone/bezoxazole analogues is provided. The present invention also provides novel [ 11 C]-labeled benzophenone/bezoxazole analogues prepared from the GMP synthesis method. Kit claims for preparing novel [ 11 C]-labeled benzophenone/bezoxazole analogues and a method of use thereof are also provided.

This application is a divisional of U.S. application number 12/301,497filed Nov. 23, 2009, which is a filing under 35 U.S.C. 371 ofinternational application number PCT/IB2007/001311, filed May 21, 2007,which claims priority to application number 60/802,416 filed May 22,2006, in The United States the entire disclosure of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a synthesis method of preparing[¹¹C]-labeled benzophenone/bezoxazole analogues. The present inventionalso relates to [¹¹C]-labeled benzophenone/bezoxazole analogues preparedfrom the synthesis method. The present invention further relates to kitsfor preparing [¹¹C]-labeled benzophenone/bezoxazole analogues as well asthe use of preparing the same. A preclinical evaluation of the labeledanalogues will be performed to verify the expected biologicalproperties.

BACKGROUND OF THE INVENTION

A group of diagnostic Positron Emission Tomography (“PET”) proceduresutilize radioactive labeled compounds, wherein the radioactive atoms arepositron emitters. Some examples of positron emitting elements includenuclides of carbon, nitrogen, or fluorine. These elements are thebackbone of almost all biological active compounds. In order to be ableto use these elements, stable isotopes are replaced with a radioactiveisotope. The radioactive labeled compounds, called tracers, aretransported, accumulated and converted exactly the same way as fornon-radioactive compounds. The PET method has possibilities to detectmalfunction on a cellular level in the investigated tissues or organs.The method is very sensitive and requires only nanomole quantities ofproduced radioactive tracers. These radioactive tracers have a half-lifein the range from 2 to 110 minutes, (e.g. ¹¹C, t_(1/2)=20.4 min). Karimiet al., Eur. J. Org. Chem., 2005, 2374-2378, Acta Upsaliensis, Uppsala2002, ISBN 91-554-5452-6 and Rahman et al., Eur. J. Org. Chem., 2004,2674-2678. Because of the radioactivity, the short half-lives and thesubmicromolar amounts of the labeled substances, extraordinary syntheticprocedures are required for the production of these tracers. The mostused precursors for the introduction of ¹¹C in a bioactive molecule are[¹¹C] iodomethane and [¹¹C] methyl triflate used in methylationreactions, [¹¹C]carbon dioxide in the Grignard reaction, and [¹¹C]carbonmonoxide in carbonylation reactions. Additionally, an important part ofthe elaboration of these procedures is the development and handling ofnew anti-inflammatory [¹¹C]-labeled tracers.

Inflammatory responses are thought to be mediated in part by theprostaglandins (“PGs”) derived from arachidonic acid by the action ofprostaglandin H synthase, which is also referred to as cyclooxygenase(“COX”). Khanum et al., Bioorg. Chem., 2004, vol. 32, 211-222.

Recent studies have shown that COX exists in two isoforms COX-1 andCOX-2. Both COX are constitutively expressed in most tissues, but COX-2,in contrast COX-1, is the mitogen inducible isoform. The inducingstimuli for COX-2 include pro-inflammatory cytokines and growth factors,implying a role for COX-2 in both inflammation and control of cellgrowth. COX isoforms are almost identical in structure but haveimportant differences in substrate and inhibitor selectivity and intheir intercellular locations. Protective PGs which preserve theintegrity of the stomach lining and maintain normal renal function in acompromised kidney, are synthesized by COX-1. In addition to theinduction of COX-2 in inflammatory lesions, it is present constitutivelyin the brain and spinal cord, where it may be involved in the nervetransmission, particularly those for pain and fever. Khanum et al.,Bioorg. Chem., 2004, vol. 32, 211-222.

COX is the principal target of nonsteroidal anti-inflammatory drugs(“NSAIDs”) and metabolites of the COX pathway are widely accepted asmediators of the inflammatory response. NSAIDs block the formation ofPGs and have anti-inflammatory, analgesic, and antipyretic activity. Thediscovery of COX-2 has made it possible to design drugs that reduceinflammation without removing the protective PGs in the stomach andkidney made by COX-1. Khanum et al., Bioorg. Chem., 2004, vol. 32,211-222.

Benzophenone analogues have been identified as potent anti-inflammatoryagents. Welstead et al. and Branacaccio et al. have reported theanti-inflammatory activity of benzoylphenylacetic acid. Khanum et al.,Bioorg. Chem., 2004, vol. 32, 211-222.

In addition, Vigorita et al. have identified polyaromatictrifluoroacetamides as anti-inflammatory agents. Vigorita et al.,Farmaco, 1989, vol. 46, 1074-1079. Accordingly, Khanum et al.synthesized hydroxybenzophenones, aroyl aryloxyacetic acid and acetamideanalogues, and evaluated them for their anti-inflammatory activity andside effects. Khanum et al., Bioorg. Chem., 2004, vol. 32, 211-222.Additionally, Unlu et al. synthesized a series of alkanoic acidderivatives and evaluated their analgesic and anti-inflammatoryactivities. Khanum et al., Bioorg. Chem., 2004, vol. 32, 211-222.

Based on the aforementioned, there is a need to find better radioactiveyields, trapping efficiency, and shorter reaction times for[¹¹C]-labeled benzophenone/benzoxazole analogues.

Furthermore, there is a need for creating inflammation imaging agentswherein these agents can be visualized by PET.

Discussion or citation of a reference herein shall not be construed asan admission that such reference is prior art to the present invention.

SUMMARY OF THE INVENTION

In view of the needs of the prior art, the present invention provides amethod of obtaining a novel series of [¹¹C]-labeledbenzophenone/benzoxazole analogues showing anti-inflammatory activitieswith the aid of positron emission tomography (“PET”). The presentinvention further provides these novel [¹¹C]-labeledbenzophenone/benzoxazole analogues. The present invention also provideskits for making novel [¹¹C]-labeled benzophenone/benzoxazole analoguesand visualizing these analogues by PET.

The [¹¹C]-labeled benzophenone/benzoxazole analogues of the presentinvention are obtained through Good Manufacturing Practice (“GMP”)syntheses. GMP is part of Quality Assurance which ensures that productsare consistently produced and controlled to the quality standardsappropriate to their intended use and as required by the MarketingAuthorization.

One embodiment of the present invention encompasses a compound offormula (I),

-   -   wherein, R₁ & R₂=alkyl, aryl, alkoxide, or a halide; R₃=H,        CH₂COOH, CH₂CONHR′, or CH₂CONR′R″ wherein R′ or R″ is alkyl,        aryl, or a combination thereof.

Another embodiment of the present invention is a compound of formula(II),

wherein R₁ & R₂=Alkyl, aryl, alkoxide, halide, or a combination thereof,and

wherein n=1, 2, or 3.

Yet an additional embodiment of the present invention is a method forpreparing compound (I), comprising the steps:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering (ThermoHypersil F2513-3, syringe filter 0.45 μm)the mixture; next adding either boronic acid or an organostannylcompound to form a compound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

A further embodiment of the invention is a method for preparing compound(II), comprising the steps:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding a halide, halide salt, or triflate to the mixture; thereafterfiltering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

Another embodiment of the present invention encompasses a kit forpreparing a compound of formula (I), wherein the kit comprises the stepsof:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

Yet a further embodiment of the present invention is a kit for preparinga compound of formula (II), wherein the kit comprises the steps of:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

DETAILED DESCRIPTION OF THE INVENTION

Due to the short half-life, low reactivity, and solubility of [¹¹C]carbon monoxide, one-pot carbonylation reactions using a micro-autoclaveis preferred, and various types of ¹¹C-carbonyl compounds have beensynthesized by this approach. In the present invention,anti-inflammatory activity of [¹¹C]-labeled benzophenone/benzoxazoleanalogues was studied. These analogues showed significantanti-inflammatory profile with low gastric ulceration incidence. Thepresent invention sets forth several additional advantages over priormethods.

The current method presents an ease of use over other methods, half thetotal synthesis and cycle time used in previous methods as well asobtaining a higher radiochemical yield when excess [¹¹C]-labeledbenzophenone/benzoxazole analogues were obtained. This excess in effectgenerated an increased trapping efficiency compared to previous methodsas well. Accordingly, the radiochemical yields of the [¹¹C]-labeledbenzophenone/benzoxazole compounds when using an organostannyl compoundwere in the range of about 50% to about 99%. The corresponding trappingefficiencies, however, were on average 22% lower than the radiochemicalyields. Furthermore, the radiochemical yields when using a boronic acidproduced about 10% lower radiochemical yield of the [¹¹C]-labeledbenzophenone/benzoxazole compounds and about 15% lower trappingefficiency. The trapping efficiency disclosed herein indicates theamount of incorporated ¹¹C in the crude product, i.e. the fraction ofradioactivity left in the crude product after purging with nitrogen gas.

Additionally, a shorter synthesis time is important for efficientlysupplying a PET center with radioactive precursor batches needed forsynthesis of tracers for PET-scans. Short synthesis times will alsoyield compounds with higher radiochemical yield and specificradioactivity (Becquerel/mole) due to less decay. Radiochemical yield(purity) is defined as the amount of radioactivity originating from aspecific substance in relation to the total amount of radioactivity in asample, expressed in %. Additionally, specific radioactivity(Becquerel/mole) is the ratio between the amount of radioactivityoriginating from a specific substance labeled with a radionuclide andthe total amount of that specific substance.

Furthermore, there is a need for a rapid efficient alternative methodfor ¹¹C-labeling of ketones owing to the variation in reportedradiochemical yields. In the present inventions method to prepare[¹¹C]-labeled benzophenone/benzoxazole analogues, a palladium complexwas used and all reactions were preformed at 125 degrees C. A reactiontime of about 2 minutes to about 5 minutes was used. The resultsindicated that the choice of analogue and solvent have a major impact onthe radiochemical yield. For instance, using DMSO as a solvent insteadof DMF increased the radiochemical yield from about 8% to about 50%.

In one embodiment of the present invention a compound of formula (I),

-   -   wherein, R₁ & R₂=alkyl, aryl, alkoxide, or a halide; R₃=H,        CH₂COOH, CH₂CONHR′, or CH₂CONR′R″ wherein R′ or R″ is alkyl,        aryl, or a combination thereof is disclosed.

In another embodiment of the present invention, a compound of formula(II),

wherein R₁ & R₂=Alkyl, aryl, alkoxide, halide, or a combination thereof,and

wherein n=1, 2, or 3 is disclosed.

Yet in a further embodiment a method for preparing compound (I),comprising the steps:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography is disclosed.

An additional embodiment of the present invention depicts a method forpreparing compound (II), comprising the steps:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding a halide, halide salt, or triflate to the mixture; thereafterfiltering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

A further embodiment depicts a method of compound (I) wherein thetris(dibenzylideneactone) palladium (0) is a catalyst.

Another embodiment of the present invention depicts a method of compound(I) wherein the halide is aryl.

Yet a further embodiment depicts a method of compound (I) wherein thetriflate is aryl.

Additionally, an embodiment of the present invention shows a method ofcompound (I), wherein the boronic acid is aromatic or aliphatic. Yet afurther embodiment shows a method of compound (I), wherein theorganostannyl compound is aliphatic.

A further embodiment discloses a method of compound (I), wherein themicroautoclave is pressurized from about 30 to about 40 MPa and areduced pressure within the vial is about 10 MPa to about 50 MPa.

Another embodiment of the present invention depicts a method of compound(II), wherein the wherein the tris(dibenzylideneactone) palladium (0) isa catalyst.

A further embodiment of the invention depicts a method according tocompound (II), wherein the halide is aryl.

An additional embodiment shows a method according to compound (II),wherein the triflate is aryl wherein the boronic acid is aromatic oraliphatic and wherein the organostannyl compound is aliphatic.

A further embodiment shows a method according to compound (II), whereinthe microautoclave is pressurized from about 30 to about 40 MPa.

Another embodiment of the present invention depicts a method accordingto compound (II), wherein the reduced pressure within the vial is about10 MPa to about 50 MPa.

Yet a further embodiment of the present invention shows a kit forpreparing a compound of formula (I), wherein the kit comprises the stepsof:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

Another embodiment of the invention shows a kit according to compound(II), wherein the tris(dibenzylideneactone) palladium (0) is a catalystand the halide is aryl.

Yet a further embodiment of the invention depicts a kit according tocompound (II), wherein the triflate is aryl and the boronic acid isaromatic or aliphatic, and the organostannyl compound is aliphatic.

Another embodiment of the invention encompasses a kit according tocompound (II), wherein the microautoclave is pressurized from about 30to about 40 MPa and the reduced pressure within the vial is about 10 MPato about 50 MPa.

Still a further embodiment of the invention depicts a kit for preparinga compound of formula (II), wherein the kit comprises the steps of:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

-   transferring the crude product to a vial at reduced pressure whereby    the crude product is then diluted with water and thereafter the    crude product is purified and identified by positron emission    tomography.

Yet an additional embodiment of the present invention depicts a kitaccording to compound (II), wherein the tris(dibenzylideneactone)palladium (0) is a catalyst.

Furthermore, an additional embodiment of the invention shows a kitaccording to compound (II), wherein the halide is aryl, the triflate isaryl, the boronic acid is aromatic or aliphatic and the organostannylcompound is aliphatic.

Yet another embodiment of the invention shows a kit according tocompound (II), wherein the microautoclave is pressurized from about 30to about 40 MPa and the reduced pressure within the vial is about 10 MPato about 50 MPa.

Another embodiment of the present invention depicts a method of use forpreparing a compound of formula (I) or formula (II),

wherein formula (I), R₁ & R₂=alkyl, aryl, alkoxide, or a halide; R₃=H,CH₂COOH, CH₂CONHR′, or CH₂CONR′R″ wherein R′ or R″ is alkyl, aryl, or acombination thereof and wherein formula (II) wherein R₁ & R₂=Alkyl,aryl, alkoxide, halide, or a combination thereof, and wherein n=1, 2, or3.

further wherein the method comprises the steps of:

flushing a solution of tris(dibenzylideneactone) palladium (0) andtri-o-tolylphosphine in

anhydrous DMSO with nitrogen to form a mixture; then

adding either a halide, halide salt, or triflate to the mixture;thereafter filtering the mixture; next

adding either boronic acid or an organostannyl compound to form acompound mixture;

then

injecting the compound mixture into a microautoclave to form a crudeproduct; finally

transferring the crude product to a vial at reduced pressure whereby thecrude product is then diluted with water and thereafter the crudeproduct is purified and identified by positron emission tomography.

Yet another embodiment of the invention shows a method of use whereinthe tris(dibenzylideneactone) palladium (0) is a catalyst.

Still a further embodiment encompasses a method of use wherein thehalide is aryl and the triflate is aryl.

A further embodiment of the invention presents a method of use whereinthe boronic acid is aromatic or aliphatic and wherein the organostannylcompound is aliphatic.

Another embodiment of the present invention shows a method of usewherein the microautoclave is pressurized from about 30 to about 40 MPaand the reduced pressure within the vial is about 10 MPa to about 50MPa.

EXAMPLE 1 Experiemental Studies

General Labeling Method

A capped vial (1 ml) containing a solution of tris(dibenzylideneactone)palladium (0) and tri-o-tolylphosphine in anhydrous DMSO (300 microL)was flushed with nitrogen. The reaction mixture was kept at roomtemperature for 10 minutes. A halide, halide salt, or a triflate,preferably an aryl triflate, was added and the resulting mixture waskept at room temperature for another 5 minutes. The reaction mixture wasfiltered (PTFE syringe filter 0.45 micrometers) before addition of aboronic acid or an organostannyl compound just before injection into themicroautoclave pre-charged with [11C]carbon monoxide. Thesemicroautoclave reactions were achieved in a stainless steel batchreactor microautoclave system (200 microL). The micro-autoclave washeated at 100 degrees C. for 5 minutes. The crude product wastransferred to a vial (3 ml) at a pressure from about 10 MPa and 50 MPa.The radioactivity was measured before and after the vial was flushedwith nitrogen. The crude product was diluted with 1.5 ml of water andpurified by semi-preparative liquid chromatography. The identity of thecompounds and radiochemical yield (purity) were established by positronemission tomography.

The following compounds were synthesized using aryl iodide and thecorresponding orgaostannyl compound. The results of the radiochemicalyield and trapping efficiency of each of these compounds are as follows:

Compound RCY TE ¹¹C-benzophenone 85% 99%¹¹C-phenyl(pyridin-3-yl)methanone 50% 99%

The two main target compounds of the present invention are depictedbelow. The halides and triflate compounds, the boronic acid compoundsand the organostannyl compounds used in the present invention.

Specific Embodiments, Citation of References

The present invention is not to be limited in scope by specificembodiments described herein. Indeed, various modifications of theinventions in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications and patent applications are cited herein, thedisclosures of which are incorporated by reference in their entireties.

What is claimed is:
 1. A compound of formula (I),

wherein, * denotes a [¹¹C], R₁ & R₂=alkyl, aryl, alkoxide, or a halide; R₃=H, CH₂COOH, CH₂CONHR′, or CH₂CONR′R″ wherein R′ or R″ is alkyl, aryl, or a combination thereof.
 2. A compound of formula (II),

wherein * denotes a [¹¹C], R₁ & R₂=Alkyl, aryl, alkoxide, halide, or a combination thereof, and wherein n =1, 2, or
 3. 3. A method for preparing compound (II) according to claim 2, comprising the steps: flushing a solution of tris(dibenzylideneactone) palladium (0) and tri-o-tolylphosphine in anhydrous DMSO with nitrogen to form a mixture; then adding an aryl halide, or aryl triflate to the mixture; thereafter filtering the mixture; next adding either aromatic or aliphatic boronic acid or an aliphatic organostannyl compound to form a compound mixture; then injecting the compound mixture into a microautoclave pre-charged with [11C] carbon monoxide to form a crude product; finally transferring the crude product to a vial at reduced pressure whereby the crude product is then diluted with water and thereafter the crude product is purified and identified by positron emission tomography.
 4. The method according to claim 3, wherein the tris(dibenzylideneactone) palladium (0) is a catalyst.
 5. The method according to claim 3, wherein the microautoclave is pressurized from about 30 to about 40 MPa.
 6. The method according to claim 3, wherein the reduced pressure within the vial is about 10 MPa to about 50 MPa. 